Intraspecific Variability in the Response of the Edible MusselMytilus chilensis (Hupe) to Ocean Acidification

Cristian Duarte & Jorge M. Navarro & Karin Acuña &

Rodrigo Torres & Patricio H. Manríquez & Marcos A. Lardies &

Cristian A. Vargas & Nelson A. Lagos & Víctor Aguilera

Received: 24 July 2013 /Revised: 28 May 2014 /Accepted: 1 June 2014# Coastal and Estuarine Research Federation 2014

Abstract Ocean acidification (OA) has been shown to affectsignificantly the net calcification process and growth rate ofmany marine calcifying organisms. Recent studies haveshown that the responses of these organisms to OA can varysignificantly among species. However, much less is knownconcerning the intraspecific variability in response to OA. Inthis study, we compared simultaneously the responses of twopopulations of the edible mussel Mytilus chilensis (Hupe)exposed to OA. Three nominal CO2 concentrations (380,700, and 1,000 μatm of CO2) were used. Negative effects ofCO2 increase on net calcification rate were only found inindividuals from Huelmo Bay. However, no effects werefound in individuals from Yaldad Bay. Moreover, OA hadnot significant effects on the shell dissolution rate in individ-uals from both localities. This suggests that the negative effectof the OA on the net calcification rate of this species is

explained by shell deposition, but not by the shell dissolutionprocesses. We do not know the specific underlying mecha-nisms responsible for these differences, but some possibilitiesare discussed. These results highlight that the responses ofmarine organism to OA can be highly variable even within thesame species. Therefore, more studies across the distributionrange of the species, considering environmental variability,are needed for a better understanding of the consequences ofOA on marine organisms. Finally, because mussels exertinfluence on their physical and biological surroundings, thenegative effects of a CO2 increase could have significantecological consequences.

Keywords Ocean acidification . Mussel . Calcification .

Growth rate

Communicated by Alberto Vieira Borges

C. Duarte (*)Departamento de Ecología y Biodiversidad, Facultad de Ecología yRecursos Naturales, Universidad Andres Bello, Santiago, Chilee-mail: cristian.duarte@unab.cl

C. Duarte :C. A. VargasCenter for the Study of Multiple-drivers on Marine Socio-EcologicalSystem (MUSELS), Universidad de Concepción, Concepción, Chile

J. M. Navarro :K. AcuñaInstituto de Ciencias Marinas y Limnológicas, Facultad de Ciencias,Universidad Austral de Chile, Valdivia, Chile

R. TorresCentro de Investigación en Ecosistemas de la Patagonia (CIEP),Coyhaique, Chile

P. H. ManríquezLaboratorio de Ecología y Conducta de la Ontogenia Temprana(LECOT), Centro de Estudios Avanzados en Zonas Áridas(CEAZA), Avenida Ossandón 877, Coquimbo, Chile

M. A. LardiesFacultad de Artes Liberales, Universidad Adolfo Ibáñez, Santiago,Chile

C. A. VargasLaboratorio de Funcionamiento de Ecosistemas Acuáticos (LAFE),Unidad de Sistemas Acuáticos, Centro de Ciencias AmbientalesEULA Chile, Universidad de Concepción, Concepción, Chile

N. A. LagosCentro de Investigación e Innovación para el Cambio Climático(CIICC), Facultad de Ciencias, Universidad Santo Tomas, Ejército146, Santiago, Chile

V. AguileraInstituto de Ciencias Naturales Alexander von Humboldt,Universidad de Antofagasta, 14 Av. Jaime Guzman,02800( P.O. Box 170, Antofagasta, Chile

Estuaries and CoastsDOI 10.1007/s12237-014-9845-y

Introduction

As a result of human activity, primarily fossil fuel combustionand cement manufacture, the atmospheric concentration ofCO2 has increased significantly during the past century(Houghton et al. 1996, 2001). About half of this anthropogen-ically produced CO2 has been absorbed by the oceans (Sabineet al. 2004). Because of the oceanic uptake of CO2, the marineconcentration of CO3

2− and the water pH are currently dis-turbed, and it is expected that in the future, these changes willbecome more pronounced (Feely et al. 2008; Wolf-Gladrowet al. 1999). For example, the pH of the ocean is already nearly0.1 unit lower than the values registered in the pre-industrialera, and it is predicted to decrease by 0.4 units by the end ofthe century and nearly 0.8 units within the next 300 years(Caldeira and Wickett 2003; Orr et al. 2005) in a processcalled ocean acidification (OA). This decrease in the pH ofthe waters will produce a reduction in the concentration ofCO3

2− (i.e., saturation state (Ω)), which could fall by about50 % by the end of the next century (e.g., Orr et al. 2005;Wolf-Gladrow et al. 1999). Both of these processes couldpotentially have a negative effect on marine calcifying organ-isms, while the reduction in the concentration of carbonateions would complicate the formation of biogenic calciumcarbonate at low levels of pH, the carbonate in already formedshells might start to dissolve, causing shells to disintegrate(e.g., Caldeira and Wickett 2003; Kleypas et al. 2006;Langdon and Atkinson 2005; Langdon et al. 2000; Leclercqet al. 2000; Orr et al. 2005; Riebesell et al. 2000 Hiebenthalet al. 2013). Additionally, increased CO2 produces hypercap-nic conditions (or acidosis), which have been shown to havenegative effects on the physiology, growth, and reproductivesuccess of marine calcifying organisms (Arnold et al. 2009;Berge et al. 2006; Cummings et al. 2011; Kurihara 2008;Michaelidis et al. 2005; Spicer et al. 2007; Widdicombe andSpicer 2008; Vhitakari et al. 2013).

Although most studies have described the negative effectsof ocean acidification, recent studies have also shown that theresponses of these organisms to the CO2 increase can varysignificantly between species (even between closely relatedspecies), with no change, or in some cases, even increasedcalcification rates in response to decreasing pH levels (due toCO2 increase) (e.g., Gooding et al. 2009; Gutowska et al.2008; Iglesias-Rodriguez et al. 2008; Miller et al. 2009; Rieset al. 2009; Parker et al. 2013). For example, laboratoryexperiments carried out with 18 marine benthic organisms(Ries et al. 2009) showed that 10 of the 18 species reducedtheir net calcification rates under elevated CO2; in sevenspecies, the net calcification increased with the intermediateand highest levels of CO2, and finally, one species (Mytilusedulis, very related to Mytilus chilensis) did not show anyresponse at all. It has been proposed that the differences in thesensitivity of the organisms and therefore the described mixed

responses might depend of the pCO2 content (or pH) to whichthey are naturally exposed in the field (Wootton et al. 2008;Hall-Spencer et al. 2008; Gazeau et al. 2010; Thomsen et al.2010, 2013). Thus, species inhabiting environments with nat-urally higher CO2 content (lower pH values) would be betteradapted to cope with the ocean acidification process(Cummings et al. 2011; Gazeau et al. 2010; Marshall et al.2008; Miller et al. 2009; Pascal et al. 2010; Widdicombe et al.2009). For example, larvae of the euryhaline oyster speciesCrassostrea ariakensis, which inhabit an environment withnaturally low pH, did not show problems in growing, calcify-ing, and developing normally when exposed to elevated CO2

levels and seawater undersaturated with respect to aragonite(Miller et al. 2009). However, it is also important to emphasizethat environments with naturally low pH may exacerbate thenegative effects of the ocean acidification (see Salisbury et al.2008; Cai et al. 2011; Melzner et al. 2013).

Overall, the studies carried out so far have shown that thereis a great need to determine whether calcifying organisms willhave the capacity to adapt to the CO2 increase in the ocean(Ilyina et al. 2009). Evaluating the intra- and not only the inter-specific variability in the responses to pH decrease will help tobetter understand this concern. Some efforts in this line havebeen made (e.g., Havenhand and Schlegel 2009; Parker et al.2011; Sunday et al. 2011; Range et al. 2012, 2013). Forexample, Parker et al. (2011), evaluating the effects of OAon the oyster Saccostrea glomerata, showed that the responseto OA may vary even within the same species. Genetic dif-ferences among populations and/or the spatial variability inthe water characteristic has been proposed as mechanisms toexplain the intraspecific variability in the responses of theorganism to OA (Parker et al. 2011; Range et al. 2011).Species of the genusMytilus inhabit a wide range of environ-mental conditions, and therefore, they are naturally exposed tohighly variable pH conditions (e.g. Vihtakari et al. 2013;Thomsen et al. 2013), which would explain in part the intra-specific variability in the response of this group to OA. InChile,M. chilensis has a wide geographic distribution (37° S–73° S) inhabiting from completely marine environments tohabitat with estuarine conditions (Krapivka et al. 2007); inconsequence, this species is naturally exposed to a wide rangeof environmental conditions. Therefore, we hypothesized thatpopulations of M. chilensis inhabiting two geographicallyseparated localities can show differential responses to OA.Therefore, the aim of this study was to compare the effect ofocean acidification on both CaCO3 net deposition and disso-lution rates and on the growth rate in juvenile individuals ofthe mussel M. chilensis (Hupe) collected from two southernChile populations.

M. chilensis is the most widely cultivated species in Chileand has been recorded (like other mussel species) as having asignificant influence on its physical and biological surround-ings (Duarte et al. 2006). Consequently, the potential negative

Estuaries and Coasts

effects produced by ocean acidification on this species couldaffect not only the ecosystem functioning but also the nationaleconomy.

Methods

Animal Collection

Juvenile individuals of M. chilensis (~1.5 cm in size and~350 mg fresh weight) were collected from culture ropes(2 m of depth) in two bays: Yaldad (43° 08′ S, 73° 44′ W)and Huelmo (41° 40′ S, 73° 02 W). Both are tidal inletslocated in southern Chile (ca. 40–42° S). Unlike HuelmoBay, Yaldad Bay receives a constant input of freshwater fromthe Yaldad river (Cáceres et al. 2008). After collection, themussels were transported in chilled conditions to the CalfucoCoastal Laboratory (Laboratorio Costero de Calfuco (LCC),ca. 39° S) and placed in plastic containers (30 cm in diameterand 40 cm in height) where they were kept in filtered(0.1 μm), aerated seawater (pH=8.1±0.01, temperature=13.1±0.01 °C, and salinity=~33 psu). To acclimatize themto experimental conditions, the mussels were kept for 2 weeksand fed daily with microalgae Isochrysis galbana (~25×106 cell/L) before the beginning of the experiments.

Experimental Setup

Seawater Acidification System and Measurements

After the acclimatization period, the experimental animalswere reared at 380, 700, and 1,000 μatm of CO2. To achievethe three different CO2 levels in the seawater (Table 1), weused laboratory-based experimental system implemented toinvestigate the consequences of ocean acidification onChilean marine calcifying organisms (Torres et al. 2013;Duarte et al. 2014; Navarro et al. 2013; Manríquez et al.2013; Vargas et al. 2013). The two highest CO2 levels

represent the worst case scenarios for the end of the years2050 and 2100 respectively (IPCC 2007; Caldeira andWickett 2003). During the experiment, the pH, temperature,salinity, and total alkalinity of the water were monitored every3 days (three replicates) in each header tank (Navarro et al.2013). The pHmeasurements were done in a closed 25-ml cellthermostatically controlled at 25.0 °C; pH was measured witha Metrohm 713 pH meter (input resistance >1013Ohm,0.1 mV sensitivity, and nominal resolution 0.001 pH units)and a glass combined double junction Ag/AgCl electrode(Metrohm model 6.0219.100) calibrated with 8.089 Tris buff-er (DOE 1994) 25 °C. pH values are therefore reported on thetotal hydrogen ion scale (DOE 1994). Temperature and salin-ity were measured using a CTD (Ocean Seven 305 Plus CTD,www.idronaut.it). Total alkalinity was measured using themethod of Haraldsson et al. (1997). The pH, AT, phosphate,dissolved silicate, and hydrographic data were used to calcu-late the remaining carbonate system parameters and the satu-ration stage of Omega Aragonite using CO2SYS software(Lewis and Wallace 1998) set with Mehrbach solubility con-stants (Mehrbach et al. 1973) readjusted by Dickson andMillero (1987).

Net Calcification Rate, Shell Dissolution, and Growth Rate

Mussels were randomly assigned to one of 15 4-L aquaria andexposed to three CO2 treatments (see above). Each treatmentwas replicated five times, and each replicate contained threeexperimental animals from each population, which were iden-tified using bee tags. In addition, two empty shells (one fromeach population) of M. chilensis, previously tagged andweighed, were placed in each container. Empty shells wereobtained by separating the soft body of live mussels, whichwere similar in size to the experimental animals. These shellswere cleaned with distilled water to eliminate any organicresidue and salt and then dried for 24 h (60 °C) beforeweighing. The experimental animals and empty shells wereselected because shell lengths, buoyant weights, and shell

Table 1 Sea water characteristics(mean±SD) used to maintainM. chilensis during the experi-mental period

TA total alkalinity, [CO32− ] car-

bonate ion concentration, Ωca

omega calcite, Ωar omegaaragonitea Based on the rate of change inpH predicted by the IS92a climatechange scenario (IPCC specialreport on emissions scenarios,http://www.grida.no/publications/other/ipcc_sr/)

CO2 system parameters Experimental treatments (nominal levels of CO2 ppm)

380 (current) 700 (year 2050a) 1,000 (year 2100a)

pH at 25 °C (pH units) 7.926±0.053 7.719±0.033 7.579±0.038

pH in situ (pH units) 8.054±0.047 7.849±0.031 7.703±0.037

Salinity (psu) 33.60 (0.41) 33.64 (0.46) 33.42 (0.64)

Temperature (°C) 16.31 (0.73) 15.98 (0.70) 15.93 (0.62)

TA (μmol Kg−1) 2251±25 2247±13 2294±39

pCO2 in situ (μatm) 388±49 664±52 979±87

[CO32−] in situ (μmol Kg−1) 161±18 106±9 79±8

Ωca 3.87±0.43 2.54±0.20 1.91±0.20

Ωar 2.49±0.28 1.63±0.13 1.23±0.13

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weights did not show significant differences among thedifferent CO2 treatments compared to the previousexperiment. During the experiments, mussels weremaintained according to Navarro et al. (2013). The aquariawere maintained without aeration and at constant temperature(13 °C). Seawater was gently changed everyday, with thecorresponding CO2 levels from the seawater acidificationsystem following the methodology proposed by Navarroet al. (2013).

The net calcification rates of the organisms under the threeCO2 treatments were estimated from changes in their buoyantweight (i.e., underwater weight) verified with dry weightmeasurements of the shells after harvesting (Palmer 1982).To prevent errors due the entrance of air within the closedvalves during the measurements of the buoyant weight (seePalmer 1982), each specimen was gently moved from therearing container to the balance. Following this procedure,only seawater remained within the valves and yields exactbuoyant weights of the experimental specimens. Buoyantweight increment is an important proxy for growth becauseit is equivalent to the calcification rates and is not affected bythe amount of seawater and tissue weight (see Palmer 1982)methodology successfully validated for M. chilensis (Duarteet al. 2014).

Themeasurements weremade on day 0 (at the beginning ofthe experiment) and at the end of a 20-day period. Shelldissolution rates, in turn, were estimated by weighing theempty shells of M. chilensis in air at the beginning and atthe end of the experiment. Live animals and empty shells wereweighed to the nearest 0.01 mg. Growth rates were estimatedfrom changes in the total body weight (to the nearest 0.01 mg)of the mussels. These measurements were made along withthose for estimating net calcification and dissolution rate. Forall measurements, each mussel was carefully removed fromthe water, immediately weighed under the water (buoyantweight), then gently blotted, and weighed in air (total weight).

Statistical Analysis

To avoid pseudoreplication problems, the variables measuredwere averaged for the three mussels from each population ineach replicate. Two-wayANOVAwas used to evaluate wheth-er CO2 levels, locality, and interaction between factors affect-ed net calcification, shell dissolution rate, growth rate (in-crease in total weight with time), and survival (Zar 1999).When the analysis showed significant interactions, a one-wayANOVA was carried out for each factor separately in eachlevel from the other factor, followed by Tukey’s a posterioriHSD test. When the analysis did not show significant interac-tions, multiple comparisons were carried out using Tukey’s aposteriori HSD test on each factor that showed significantdifferences (Underwood 1997). Net calcification rate and totalweight increase were expressed in milligrams per mussel per

day, while shell dissolution was expressed in micrograms permussel per day. All analyses were carried out using Statisticaversion 7.0. Assumptions of normality and homoscedasticityfor the ANOVA’s test were evaluated using the Kolmogorov-Smirnov and Bartlett tests, respectively (Zar 1999).

Results

Water Characteristics

The measured parameters of the seawater are shown inTable 1. Averaging all measurements, the mean (±SD) pHvalues of the treatments (at 25 °C) were 7.98±0.05, 7.56±0.005, and 7.47±0.05 corresponding to the CO2 treatments at380, 700, and 1,000 μatm, respectively (Table 1). The carbon-ate contents decreased with increase in CO2, but the seawaterwas not undersaturated with respect to aragonite and calcite inany of the treatments.

Net Calcification and Shell Dissolution

In specimens from Huelmo Bay, the net calcification (estimat-ed by changes in the buoyant weight) was significantly higherat the control CO2 treatment than in the intermediate andhighest CO2 treatments, while in this same bay, the net calci-fication recorded at the intermediate CO2 concentration wasnot significantly different from the highest CO2 treatment(two-way ANOVA; locality: F1,24=8.71, p<0.05; CO2 con-centration: F2,24=0.55, p>0.05; locality × CO2 concentration:F2,18=5.08, p<0.05; one-way ANOVA: F2,12=6.22, p<0.05;Fig. 1). No significant effect of CO2 concentrations on netcalcification was recorded in individuals from Yaldad Bay

Fig. 1 Net calcification rate (mean±SD) of the individuals ofM. chilensisfrom the bays of Yaldad and Huelmo reared under three nominal CO2

levels (Table 1)

Estuaries and Coasts

(one-way ANOVA: F2,12=1.05, p>0.05). There was a loca-tion effect, which was driven by higher calcification rate incontrol animals from Huelmo Bay.

Shell dissolution of M. chilensis (estimated by changes inempty shell weight) collected in the different localities andCO2 treatments are shown in Fig. 2. The shell dissolution rateover the studied period was not affected by CO2 concentra-tions. However, a locality effect was detected with values ofshell dissolution significantly higher in individuals fromHuelmo than from Yaldad Bay (two-way ANOVA; locality:F1,24=44.19, p<0.05; CO2 concentration: F2,24=1.11,p>0.05; locality × CO2 concentration: F2,24=0.54, p>0.05;Fig. 2).

Total weight increase (growth rate) under the different CO2

levels over the duration of the experiment is shown in Fig. 3.Total weight increase of bivalves was significantly higher inindividuals from Huelmo Bay. The CO2 concentration alsoaffected, significantly and negatively, this parameter, so totalweight increase was lower in the highest CO2 concentrationthan in the control treatment, while total weight increaseregistered at intermediate CO2 concentration was not signifi-cantly different from the other two treatments (two-wayANOVA; locality: F1,24=31.70, p<0.05; CO2 concentration:F2,24=4.88, p<0.05; locality×CO2 concentration: F2,24=1.97, p>0.05; Fig. 3).

Discussion

The short period of exposure used in our study (20 days) wasenough to detect significant effects of OA on the net calcifi-cation and growth rate of juvenile mussels of M. chilensis;such had been shown in a previous study (see Nienhuis et al.

2010), where short (even shorter than this study) exposuretime allowed to detect OA acidification effects on marinemollusk. In addition, it is important to emphasize that in aprevious study, the responses of this species when exposed fora longer time to high CO2 levels (60 days; Duarte et al. 2014)were similar to those detected in the present study. Thissuggests that OA might have significant effects on net calci-fication in a short period of time.

Although the studies carried out to evaluate OA effectshave used different methodologies, increasing evidence sug-gests that the effects of the projected CO2 increase on calcifi-cation and growth rates are species-specific, being in somecases negative (e.g., Duarte et al. 2014; Miller et al. 2009;Nienhuis et al. 2010), in others positive (e.g. Findlay et al.2009; Iglesias-Rodriguez et al. 2008; Gooding et al. 2009;Gutowska et al. 2010), and in others neutral (e.g. Fabry et al.2008; Range et al. 2011; Ries et al. 2009). These contrastingresponses have also been registered within species closelyrelated to M. chilensis (i.e., Mytilid species) (Berge et al.2006; Gazeau et al. 2007; Michaelidis et al. 2005; Rangeet al. 2012, 2013, 2013; Thomsen and Melzner 2010;Vhitakari et al. 2013). In this study, we show thatM. chilensis shows substantial intraspecific variation in re-sponse to ocean acidification. Our experiments showed thatincreased concentration of CO2 affects negatively the netcalcification and growth rate of M. chilensis, as it had previ-ously been registered in this species (Duarte et al. 2014).However, in the case of the net calcification rate, only theindividuals from Huelmo Bay were affected by OA, but notthose of Yaldad Bay for which no significant differences incalcification rate were found among the three CO2 treatments.It should be noted that the absence of mortality during theexperiment indicated that the projected CO2 levels had chron-ic, but not lethal, effects on those animals.

Fig. 2 Dissolution rate (mean±SD) of the individuals of M. chilensisfrom the bays of Yaldad and Huelmo reared under three nominal CO2

levels (Table 1)

Fig. 3 Total body weight increase (mean±SD) of the individuals ofM. chilensis from the bays of Yaldad and Huelmo reared under threenominal CO2 levels (Table 1)

Estuaries and Coasts

Many coastal organisms may already be exposed to waterswith high CO2 levels and subsaturating levels of CO3

2−,similar to those projected to occur in the next century(Talmage and Gobler 2009; Wootton et al. 2008; Hall-Spencer et al. 2008; Pansch et al. 2013; Thomsen et al.2010, 2013). Recent studies suggest that taxa associated withenvironments that naturally present high CO2 levels may havephysiological and metabolic adaptations and consequently tobe better acclimatized to ocean acidification (e.g., Cummingset al. 2011; Findlay et al. 2009; Kurihara 2008; Pascal et al.2010; Widdicombe et al. 2009; Sunday et al. 2011). Forexample, the expression of the chitin synthase (CHS) enzyme,which is key in the synthesis of bivalve shells, was upregulat-ed in individuals of the Antarctic bivalve Laternula ellipticaexposed to hypercapnic conditions, indicating some degree ofadaptation to ocean acidification in this species (Cummingset al. 2011). Similarly, Pascal et al. (2010) recorded thatcopepods associated with sediments with higher CO2 concen-trations were better adapted to hypercapnic environments thancopepods inhabiting sediments with lower CO2 levels. In thiscontext, the lower susceptibility of the net calcification toocean acidification recorded in the Yaldad Bay mussels couldhave resulted from an adaptation of these organisms to thefreshwater inputs (from Yaldad River), which may producenaturally lower pH values and low carbonate ion contents (seeFeely et al. 2008; Salisbury et al. 2008), as those to which theorganisms were experimentally exposed. Our preliminary datawould give support to this hypothesis since the pH and theCaCO3 saturation state of water column where mussels arecultured in Yaldad Bay may be substantially lower (pH=25–7.4, Omega Ar <1) compared to Huelmo Bay (pH=25–8.1,Omega Ar >1). We have no evidence to conclude that localadaptation to freshwater inputs at Yaldad River is the ultimatecause for the reported differences. Therefore, we propose thisexplanation just as a potential mechanism to explain thereported different responses between both populations. Morestudies comparing the water column characteristics betweenboth bays are needed to support this hypothesis.

A very striking result of this research was that the YaldadBay individuals presented to be independent on the CO2 levelsand lower values for the net calcification and growth rate thanthose recorded in the Huelmo Bay individuals. In other bi-valve species, it has been registered that inhabiting in areaswith constant freshwater inputs could have a cost for theseorganisms. For example, Salisbury et al. (2008) recorded thatthe discharges of the Kennebec River into the Gulf of Maine,which are more acidic than the receptor body, have thepotential to negatively affect the survival and settlement ofthe commercially valuable clam Mya arenaria. Similarly,Tunnicliffe et al. (2009) found that populations of the ventmussel Bathymodiolus brevior, living in areas with naturallyhigh CO2 levels and low pH, presented thinner shells andlower daily growth than populations of this species living in

waters with pH>7.8. This could be the case for the individualsof Yaldad Bay, although it is necessary, again, to indicate thatmore data are needed to confirm this (see above). It must beindicated that the lower calcification and growth rate of theYaldad Bay individuals could also result from natural intra-specific variability between the two populations. However,independent on the factors that account for these differences(out of the scope of this study), the results highlight theimportance of considering the intraspecific variability in theresponses of the individuals to OA, as previously demonstrat-ed (Range et al. 2013). We strongly agree that laboratorysettings may impose limitations since those conditions donot recreate the natural environment. However, in our study,individuals of both populations were exposed to similar labo-ratory conditions.

Animal net carbonate production is influenced by shelldeposition and shell dissolution (Nienhuis et al. 2010).Consequently, both of these processes must be evaluatedwhen the effects of ocean acidification on the net calcificationrate are studied (Iglesias-Rodriguez et al. 2008; Nienhuis et al.2010) since an increase in the deposition rate in order toovercome shell dissolution could disguise the effects of oceanacidification (Iglesias-Rodriguez et al. 2008). The shell disso-lution rate of the individuals of both populations did notpresent significant differences among the three CO2 treat-ments, which would indicate two things: (i) the Yaldad Bayindividuals did not have to increase their deposition rate tocompensate for shell dissolution, thus confirming that oceanacidification did not affect the net calcification rate of thispopulation at all and (ii) the net calcification rate of theHuelmo Bay individuals was not affected by this process,but by the deposition process. This result disagrees with thestudy of Nienhuis et al. (2010), where elevated CO2 concen-tration affected the shell dissolution rate but not the depositionrate in the intertidal snail Nucella lamellose. It is important toemphasize that in both populations, the shell dissolutionaccounted less than 6 % of the total shell weight, and thatthe experimental seawater was not undersaturated for calciteor aragonite. Therefore, the reported shell lost could be aminor artifact that contributed equally to each treatment andassociated with handling during the measurements.Interestingly, this reduction in shell calcification of theHuelmo Bay population was found despite the fact that theseawater in the treatments with higher CO2 content was satu-rated with respect to the calcium carbonate measured.Different studies have reported that the calcification ratemay decrease even if seawater remains above the saturationstate (e.g., Beniash et al. 2010; Ries et al. 2009; Talmage andGobler 2009). For example, in a recent study, Beniash et al.(2010) showed that exposure to high CO2 levels resulted in asignificant reduction in the shell deposition rate of the oysterCrassostrea virginica, and that this reduction occurred in spiteof the fact that the seawater was not undersaturated with

Estuaries and Coasts

respect to calcite. Similarly, another study also reported asignificant decrease in larval growth (total length) of thebivalves Mercenaria mercenaria and Argopecten irradianswhen exposed to high CO2 levels even in seawater saturatedwith respect to calcite (Talmage and Gobler 2009). The calci-fication rates of the mussel M. edulis and the oysterCrassostrea gigas decreased in seawater enriched with CO2,but not supersaturated with respect to calcium carbonate(Gazeau et al. 2007). The calcification rate of other groups,such as foraminifera, coccolithophores, and corals, has alsobeen shown to decrease with low carbonate concentrationeven in saturated seawaters (Kleypas et al. 2006; Riebesellet al. 2000). In fact, Doney et al. (2009) indicated that formany organisms, the chemical saturation threshold do notrelate with the calcification thresholds. Therefore, the supplyof carbonates would not explain the negative effects on the netcalcification rate of the Huelmo Bay individuals recorded inthis study; these calcification problems would therefore mostlikely be related with hypercapnia or acidosis; this has previ-ously been proposed by Arnold et al. (2009) to explain whythe lobster Homarus gammarus showed a reduction in themineral content (calcium and magnesium) of its carapace intreatments with CO2 acidified sea water even though in thesetreatments, the seawater was not undersaturated with respectto the calcium carbonate polymorphs measured by theauthors.

Conclusions

This study evaluated simultaneously and under similar condi-tions the impacts of OA on individuals from two populationsof the same species over a 20-day time scale. The results ofthis study corroborate the increasing evidence that shows thatsubstantial intraspecific variation in response to increments ofCO2 levels may also occur, which warns the danger of gener-alizing the results obtained evenwithin the same species. Onlyin the higher CO2 concentrations that the responses ofM. chilensis were negatively affected, which suggests that,within certain limits, this species can cope with some CO2

increase. The absence of mortality indicated that the projectedCO2 levels had chronic, but not lethal, effects on the experi-mental animals.More studies should be focused on comparingdirectly the responses of different populations to OA and thespecific mechanisms (e.g., physiological adaptation) that al-low some populations to be less sensitive to this phenomenon.

Acknowledgments The authors are grateful to Jorge López, BárbaraCisternas, María Elisa Jara, and Loreto Mardones for their valuableassistance during the experiments and for the algae production. Themanuscript was improved thanks to the comments from three anonymousreviewers. Financial support provided by the Programa de InvestigaciónAsociativa, PIA-CONICYT-CHILE (Grant Anillos ACT-132) is grate-fully acknowledged. This is Anillos ACT-132 publication no. 9. This

study was also partially funded by Project Fondecyt no. 11110407 (toCD). The Millennium Scientific 13 Initiative Grant IC120019 also sup-ported this work during the final stage. CAV is supported by Red 14Doctoral REDOC.CTA, MINEDUC project UCO1202 at U. deConcepción.

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